Mitochondria are the sole energy-producing organelles in all eukaryotic cells, and therefore play a critical role in maintaining proper cellular bioenergetics, homeostatic levels and cellular life cycles. Similarly, chloroplasts are also efficient ATP-producing machines that use light as the source of energy rather than sugars or fatty acids. Both mitochondria and chloroplasts contain multiple copies of organelle DNA that is replicated and transcribed in the organelles. In mammals, mitochondrial DNA (mtDNA) is a circular, approximately 16.5 kilobase, intronless genome that encodes 13 electron transport chain (ETC) proteins, 2 ribosomal RNA's and 22 tRNA's. Most insights into mitochondrial genetics have come in yeast, where biolistic transformation allows for engineering of mitochondrial replicons. However, many features of mammalian mitochondrial gene expression and respiratory chain biogenesis are not reproducible in yeast.
In mammals, cytoplasmic fusion and microinjection are used to introduce donor mitochondria, but these techniques fail to provide a mechanism for the direct manipulation of mtDNA. In addition the uptake of exogenous DNA into mitochondria involving the protein import pathway has been reported from two laboratories. Vestweber and Schatz ([1989] Nature (London) 338:170-172) achieved uptake of a 24 bp both single-and double-stranded oligonucleotide into yeast mitochondria by coupling the 5′ end of the oligonucleotide to a precursor protein consisting of the yeast cytochrome c oxidase subunit IV presequence fused to a modified mouse dihydrofolate reductase. More recently, Seibel et al. (1995, Nucleic Acids Research 23:10-17) reported the import into the mitochondrial matrix of double-stranded DNA molecules conjugated to the amino-terminal leader peptide of the rat ornithine-transcarbamylase. Both studies, however, were done with isolated mitochondria, not addressing the question of how oligonucleotide-peptideconjugates will pass the cytosolic membrane and reach mitochondrial proximity. U.S. Pat. No. 6,171,863 discloses the use of dequalinium-DNA complexes as a vehicle for delivering DNA to the interior of cells and potentially to the mitochondria Because the DNA is associated with dequalinium, the resulting complex has a positive charge. The positively charged complex is attracted to negatively charged compartments. Thus, US Pat. No. 6,171,863 discloses delivery of DNA to negatively charged compartments, and does not disclose the specific delivery DNA to mitochondria or chloroplasts. Indeed, no technique has been disclosed for targeting specific organelles, for example the chloroplast or mitochondria, for the delivery of nucleic acids using a receptor:ligand mechanism.
Thus the inability to specifically manipulate the chloroplast and mitochondrial genome has hampered researchers' efforts to fully understand chloroplast and mtDNA replication, transcription, and translation processes. The ability to specifically manipulate mtDNA and introduce it into living cells would greatly enhance researchers'ability to fully investigate the function of individual chloroplast/mitochondrial genes and overall chloroplast/mitochondrial function.
Furthermore, the ability to manipulate the mitochondrial genome also provides a novel method of treating diseases associated with defective mitochondrial function. With age, the function of mitochondria decreases with a marked increase of mutations and large deletions of mtDNA. In particular, oxidative damage increases with age, often leading to a higher rate of mtDNA mutations. Aside from known mtDNA mutations, several forms of cancer and neurodegeneration are associated with mutations in mtDNA. For example, mutations in mitochondrial DNA are the suspected cause of a host of degenerative neurological diseases including Alzheimers, Parkinsons and adult-onset diabetes. These mutations result in decreased electron transport chain efficiency, and the build-up of mtDNA deletions due to free radical damage (aging).
In addition, given the bioenergetic functions of chloroplasts, the ability to introduce exogenous genes or otherwise manipulate the chloroplast genome could have a tremendous impact on increasing the vitality and yields of crops and other plants. For example, introduction of genes into chloroplast may lead to plants with increased viability in otherwise hostile environments and increased efficiency of photosynthesis. In addition, the expression of exogenous genes within the chloroplasts is believed to be significantly more efficient in chloroplasts relative the expression of exogenous genes introduced into the nucleus of the cell. Thus transfection of chloroplasts may allow for more effective biosynthesis strategies for commercial compounds.
Phylogenetically, mitochondria and chloroplasts resemble early bacteria As such, applicants recognized that bacterial viruses (e.g., bacteriophage lambda), could potentially be utilized to introduce DNA into these animal and plant organelles.